Identifying the regulatory regions of the genome is critical for understanding the regulatory networks underlying human development and disease. Chromatin immunoprecipitation (ChIP) coupled with microarray (ChIP-chip) or sequencing (ChIP- seq) is a widely used technique for understanding the genome-wide binding pattern of regulatory proteins. A key bottleneck hindering broader application of the Chip approaches is the lack of ChIP-grade antibodies against transcription factors. The use of polyclonal antibodies, monoclonal antibodies and epitope-tagging approaches in ChIP has issues with cost, speed or performance that limit their use in large scale applications. The long term goal of this project is to develop a high performance, low cost substitute for traditional antibodies. The objective of this proposal is to select an immunoglobulin- like scaffold with properties that antibody substitutes derived from this scaffold. The central hypothesis underlying this work is the 10-15 kD immunoglobulin-like scaffolds, screened and produced in E. coli, can serve as a low cost substitute for full length immunoglobulins in ChIP experiments. Several groups have shown that loops on immunoglobulin domains can be modified to generate complementarity determining regions specific for a ligand of interest. Preliminary results with a novel, candidate scaffold have demonstrated that it is highly expressed in E. coli, tolerates the insertion of loops with randomized amino acids, and is compatible with a bacterial display technology that we have licensed. The specific aims of this proposal are: 1) identify a protein scaffold that is suitable for the production of ChIP-grade antibody substitutes and 2) select a scaffold-compatible display system suitable for high- throughput, low cost screening for ChIP-grade antibodies, and generate binders to transcription factors well characterized in ChIP studies, such as neuron-restrictive silencer factor (NRSF). This project is innovative in applying approaches previously established with therapeutic antibody mimetics to the novel application of developing low cost, high performance antibody substitutes for ChIP. This innovative approach is expected to establish a system with the potential for the routine, rapid, low cost production of antibody substitutes whose selectivity and affinity are comparable to monoclonal antibodies. The availability of these antibody substitutes will enable ChIP studies with a broader range of transcription factors in support of ENCODE (the Encyclopedia of DNA Elements) and other genomics initiatives. Furthermore, these antibody substitutes are likely to have a broader positive impact when applied to other large- scale, post-genomic projects, such as genome-wide immunoprecipitation or subcellular localization surveys. PUBLIC HEALTH RELEVANCE: Understanding the regulatory regions of the genome is critical for understanding the regulatory networks underlying human development and disease. The use of chromatin immunoprecipitation, a key technique for genome-wide efforts to map regulatory regions, is limited by the scarcity of appropriate antibody tools. The goal of this project is to develop a system to generate low cost, high performance antibody substitutes to enable larger scale mapping studies with chromatin immunoprecipitation.